PERCUTANEOUS ABSORPTION 491 Although 'disappearance measurements' are essential in order to obtain direct evidence of absorption of the test substance from the site of topical application, some idea of the rate of absorption may be obtained by measur- ing its rate of excretion in urine or faeces, from its deposition in internal organs, or by observing some known biological effect as a consequence of absorption. The list of compounds studied in this way in both animals and humans is an exhaustive one and only representative examples can be mentioned here. The absorption of mercury from intact and abraded skin was investi- gated critically by Sorby and Plein (70) by the use of •'øaHg. These workers used ammoniated mercury labelled with 2øaHg applied to the skin in the form of an ointment, and, 24 h later, the kidneys were removed for the deter- mination of mercury content since after absorption the metal accumulates chiefly in this organ (77). Estimation of the radio-active label in the urine has been found useful in other investigations especially in the investigation of the percutaneous absorption of steroids (67). The radio-active label, in this series of investigations, was found useful not only in determining the rate of absorption but in rendering unnecessay the elaborate analytical procedures required for identifying and measuring the metabolites. In other experiments, the radio-active label has been found useful in assisting the isolation and identification of the metabolites. For example, the presence of a•S in sodium pyridinethione was found to be of considerable assistance in the identification of its metabolite, pyridine-N-oxide-2-sulphonic acid in the urine of rats after dermal application of the parent compound (78). The pharmacological or other known biological effects of a particular compound can sometimes be used to investigate percutaneous absorption. Topical application of microgram quantities of steroids incorporated in a cream or ointment base produce local vasoconstriction which is visible as blanching. The degree and extent of blanching by topical corticosteroids was suggested as an indicator of percutaneous absorption and as a means of comparing absorption and efficacy in tests for new steroids (79, 80). Further experience indicated that this method of testing was neither accurate nor reproducible the degree of blanching was subject to 'observer error' and was found to vary in the same individual at different times of day even though the same anatomical site was used. The surrounding vascular skin colour and degree of pigmentation were found to interfere considerably with the interpretation of results. Dissolving the steriod in ethyl alcohol did not appreciably improve the reproducibility of the vasoconstriction (81). Despite these limitations, this method gave a reasonably close approximation

492 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS between vasoconstrictor ability and clinical efficiency (82-84). The produc- tion of an area of anaesthesia by a topically applied substance could be used as a means of detecting percutaneous absorption, but it is even more subject to error than the vasoconstriction test (85). Other pharmacological parameters have been found useful in determin- ing percutaneous absorption and are still used occasionally in order to relate pharmacological action with rates of absorption measured by other tests. For example, changes in serum cholinesterase have been used to compare the toxicity of parathion and paraoxon after dermal application (54, 86, 87). Other criteria which have been used occasionally are death of test animals (so-called cutaneous LD50) from topically applied compounds or organ damage assessed histologically. Such an approach does not give an accurate measurement of percutaneous absorption but may be useful in order to obtain data on the dose levels at which certain compounds may produce systemic toxicity when applied topically. This approach was used by Wahlberg (74) in a comparative study of the systemic toxicity of mercuric chloride, cobaltous chloride and sodium chromate and is extensively used in determining the dermal toxicity of pesticides (88). It is not always possible to compare the results of percutaneous absorp- tion using an isotope-labelled compound with those obtained using other methods of measurement. In many instances the sensitivity of the analytical techniques employed is very much less than the radio-isotope techniques so that meaningful comparisons are difficult. Antibiotic assays using micro- biological techniques are sensitive and accurate and Vickers (89) compared the percutaneous absorption of sodium fusidate and fusidic acid, using such techniques with the result of absorption obtained by standard •4C-labelling techniques. Both by in vivo and in vitro methods, the results were found to be very close confirming the reliability of the radio-isotope techniques. The measurement or demonstration of skin absorption using biological effects is limited to compounds having a high biological activity. The use of this technique for cosmetic ingredients is therefore limited but is im- portant in the case of biologically active materials used in some permanent waving solutions or that will control bacteria on the skin, influence meta- bolism in such ways as to improve the texture or appearance of the skin, retard perspiration, or control dandruff. Autoradiography Autoradiography has been employed in the study of percutaneous